Abstract Pseudomonas aeruginosa (PA) is the bacterium most frequently isolated from the respiratory tract of ICU pa- tients in the US and is a major cause of pneumonia in intubated patients. Furthermore, infection with PA gener- ally has a poor prognosis, with an estimated 40-69% of PA ventilator-associated pneumonia (VAP) cases re- sulting in mortality, and more than 30% of survivors suffer recurrence following standard-of-care antimicrobial therapy. The overall goal of this project is to address this critical medical need by discovering novel small mole- cule inhibitors of the type-three secretion system (T3SS) translocon comprised of PopB & PopD in the mam- malian cell membrane and developing them into new therapeutic agents against PA. The T3SS is the major virulence factor contributing to the establishment and dissemination of PA infections, and its presence is asso- ciated with poor clinical outcomes and death in infected patients. The strategy of this project is to administer T3SS inhibitors to PA pneumonia patients as adjunctive agents to enhance the function of standard-of-care antibiotics by enabling phagocytic cells to eliminate persisters and antibiotic-resistant bacteria. Such agents will by-pass the pathogen intrinsic resistance mechanisms -- a poorly permeable outer membrane and multiple ef- flux pumps. The approach of this proposal is to identify small molecule inhibitors of the PopB/PopD translocon assembly in cell membranes. In preliminary studies, the following was demonstrated: (a) PopB and PopD form a hetero-dimer in model lipid bilayers, (b) PopB assists the insertion of PopD into the membrane of cultured cells, and (c) only functional hetero-oligomers expose the N-terminus of PopD to the host cytosol. Conse- quently, exposure of the PopD N-terminus to the host cytosol will be used as a reporter of accurate T3SS translocon assembly. Self-complementation of a split green fluorescent protein (GFP) will be used to detect properly inserted PopD. A truncated optimized superfolder-GFP (GFP1-10) missing a 16 amino acid residue ?- strand (GFP11), will be stably expressed in host mammalian cells. The missing GFP11 strand has been added to the N-terminus of PopD as an epitope label. Assembly of functional translocons will expose the GFP11 epitope on PopD to the host cell cytosol where it will complement GFP1-10 and be detected by fluorescence. Our studies have already demonstrated that GFP11-PopD is active for effector translocation when added to a PA?popD strain. In Phase I, we will develop this fluorescent cellular HTS assay for inhibitors of T3SS trans- locon assembly. Compounds will be screened and hits confirmed and validated with secondary assays and counter-screens. Compounds that meet the assay funnel criteria will be prioritized for chemical optimization and in vivo proof efficacy in Phase II. Specific Aims are: (1) Complete development of cellular screens to iden- tify inhibitors of the T3SS translocon assembly (Yr 1); (2) Optimize the T3SS translocon assembly screen for HTS, apply it to libraries of diverse compounds, and confirm inhibitors (Yr 1-2); (3) Validate T3SS translocon assembly inhibitors to determine potency, selectivity, preliminary SAR, and predicted ADME properties (Yr 2).